Vitrification of In Vitro‐produced Bovine Embryos by Addition of Ethylene Glycol in One‐step

Contents The objective of this study was to simplify two‐step addition of cryoprotectant for vitrification of bovine embryos by developing a one‐step procedure. Survival was calculated as a percentage of non‐vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one‐ or two‐step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one‐step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two‐step addition (85% vs 98%, respectively). Based on these results, experiments 2–4 were designed to test one‐step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 m ethylene glycol for 2.5 or 3.5 min. At 24 h post‐vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 m 38%, 7 m 51%, 8 m 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 m ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p < 0.05). Experiment 4 concerned one‐step addition of cryoprotectant to day 6 bovine morulae, exposed to 7 m ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one‐step addition of cryoprotectant, but others do not. Although, one‐step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro ‐produced bovine embryos.